The prevalence of diabetes (DM) worldwide has soared above 380 million individuals. The primary causes of morbidity and mortality in these patients are diabetes-related macrovascular and microvascular disease. DM vascular disease has critical pathophysiological differences from vascular disease seen in non-DM patients. Currently therapies to combat vascular disease are significantly less effective in DM patients compared to non-DM patients. Novel therapies targeted at disrupting pathophysiological pathways of particular importance in DM vascular disease may offer significant benefits for the reduction of adverse vascular events in DM. DM vascular disease begins with the development of vascular endothelial dysfunction-a state characterized by increased vascular inflammation and increased vasoconstrictive and pro-thrombotic tendencies. In DM, we and others have discovered endothelial dysfunction can be initiated by critical changes in endothelial mitochondrial function occurring secondary to excessive mitochondrial fission. These changes appear both following acute exposure to abnormal glucose as well as being evident during the chronic abnormal glucose exposures of DM. Our preliminary data suggest both acute impairment of endothelial function by high or low glucose exposure and chronic DM endothelial dysfunction occur through a common mechanism-the activation and binding of dynamin-related protein-1 (Drp1), a cytosolic-based GTPase enzyme, to docking proteins located on the outer mitochondrial membrane. This binding initiates excessive mitochondrial fission and triggers mitochondrial and endothelial dysfunction. Further, our preliminary data strongly suggest Fis1 is the critical Drp1 docking protein in this process. This application employs an innovative translational approach that uniquely combines critical pharmacological and molecular studies targeting the Drp1-Fis1 interaction in intact human vessels and endothelial cells from human subjects with structure-based drug design and testing of resulting compounds in relevant patient-derived tissues. Our approach holds great promise to lead directly to identifying a first-i-class pharmacological agent that could significantly reduce heart attacks, strokes, peripheral vascular disease, renal disease, blindness, and neuropathy in the world's nearly 400 million cases of diabetes. In Aim 1, we will determine whether acute in vivo exposure to high or low glucose levels induces mitochondrial fission and excess mitochondrial reactive oxygen species production. Further, we will determine whether impairment of endothelium-dependent vasodilation and nitric oxide (NO) bioavailability in intact arterioles from DM patients is Drp1 and/or Fis1-dependent manner. In Aim 2, we will determine whether the chronic impairment of endothelium-dependent vasodilation and NO bioavailability in intact arterioles from human with DM can be reversed by suppression of Fis1 and/or Drp1 expression. In Aim 3, we will identify small molecules to that specifically disrupt the Drp1-Fis1 interaction, validate these findings, an test the efficacy these small molecules on ex vivo human arterioles from DM subjects.